Despite continued interest in novel and repurposed small molecule inhibitors, the standard of care for adult patients diagnosed with de novo acute myeloid leukemia (AML) has remained largely unchanged over the last three decades. Recent developments have shed light on the dependence of AML cells on the antiapoptotic protein BCL-2, revealing a molecular dependency that can be exploited. Concurrently, a class of targeted therapies, termed BH3 mimetics, have been developed for the purpose of inhibiting antiapoptotic BCL-2 family proteins. One of these agents, ABT-199, is a specific inhibitor of BCL-2 and is moving into phase III clinical trials for treatment of AML. Given that all targeted therapies to date are subject to resistance, it is not surprising that AML cells are capable of acquiring resistance to ABT-199. Recently, we used a pathway-centric, gain-of-function screen to nominate MCL-1 and BCL-XL as major drivers of resistance to ABT-199 in AML. Further, we demonstrated that one or both proteins were upregulated in six independent ABT-199-resistant AML cell lines derived through chronic drug exposure and that co-targeting these proteins with ABT-199 can delay or even forestall the onset of acquired resistance. Presently, however, our ability to directly target these proteins is limited by the lack of potent and orally bioavailable compounds. In the proposed work, we will use both pharmacological, candidate-based methods and unbiased, CRISPR/Cas9-based screens to identify therapeutic targets that enhance the activity of ABT-199 in AML through their inhibition of BCL-XL and MCL-1. Hits from these assays will be credentialed through a series of computational and in vitro approaches to identify those combinations with greatest translational potential, and these lead combinations will subsequently be studied in an in vivo murine model of AML. Together, the studies proposed here promise to identify new combination therapies that efficiently target apoptosis in AML with the goal of leading to more penetrant and durable therapies for patients